Fluorescent proteins (FPs) make it possible to visualize biological processes through in vivo imaging and in vitro fluorescence labeling. Processes like protein expression, localization, degradation, and interaction can be observed through fusion of a protein of interest with a FP. Green Fluorescent Protein (GFP) from Aequorea victoria and its varied derivatives constitute a multi-colored toolbox ranging from blue to yellow, with red-shifted FPs (RFPs) originating mostly from the sea anemone Discosoma striata (Day and Davidson, 2009). Following the discovery and subsequent explosion of available FPs, came the development of their use in techniques like Fluorescence or Förster Resonance Energy Transfer (FRET), Bioluminescence Resonance Energy Transfer (BRET), and Bimolecular Fluorescence Complementation (BiFC), all of which capitalize on the variety of choices in excitation and emission maxima characteristic to each FP.
In GFP-like and RFP-like fluorophores, fluorescence emanates from a chromophore developed by the formation of an imidazolinone ring system between two centrally located residues. However, complete maturation of the chromophore requires oxidation of an adjacent tyrosine residue, making molecular oxygen a strict requirement for these systems (Tsien, 1998). For example, the chromophore of GFP itself is formed by the cyclization of the tripeptide Ser65-Tyr66-Gly67.
Bacterial MurB enzymes are a family of flavoproteins that non-covalently bind flavin adenine dinucleotide (FAD). The MurB enzyme family catalyzes a step in peptidoglycan biosynthesis. Because peptidoglycans are cell wall components, enzymes in this pathway have been targets for developing antimicrobial compounds. In a physiological setting, MurB flavoprotein enzymes catalyze a hydride transfer from NADPH to the substrate through FAD to produce the final reduced product, UDP-N-acetylmuramic acid (El Zoelby et al., 2003). A byproduct of FAD binding is fluorescence due to FAD's intrinsic fluorescent properties. Free FAD fluorescence is rather weak (ΦF=0.032) due the quenching effects of the adenine moiety. However, sequestration within a protein environment can enhance its fluorescence (Munro and Noble, 1999).